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A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI-SNF complex component

ABSTRACT: Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI-SNF family of chromatin remodelling complexes in an NCAPH2-independent manner.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD022620 | Pride | 2020-12-14

REPOSITORIES: Pride

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A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

Project description:Condensin complexes compact and disentangle chromosomes in preparation for cell division. Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI/SNF family of chromatin remodelling complexes in an NCAPH2-independent manner. This cross-reactivity with an abundant chromatin-associated factor is likely to affect the interpretation of protein and chromatin immunoprecipitation experiments that make use of this antibody probe.

2020-12-14 | PXD022664 | Pride

Condensin association with chromosomes depends on a DNA binding domain formed by its HEAT-repeat subunits

Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensinM-bM-^@M-^Ys HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain. Analysis of condensin binding genomewide in a wild type and a condensin mutant

2014-05-16 | E-GEOD-55948 | biostudies-arrayexpress

Condensin association with chromosomes depends on a DNA binding domain formed by its HEAT-repeat subunits

Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensin’s HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain.

2014-05-16 | GSE55948 | GEO

Condensin II inactivation in interphase does not affect chromatin folding or gene expression

Project description:Condensin complexes have been proposed to play a prominent role in interphase chromatin organization and control of gene expression. Here, we report that the deletion of the central condensin II kleisin subunit Ncaph2 in differentiated mouse hepatocytes does not lead to significant changes in chromosome organization or in gene expression. Both observations challenge current views that implicate condensin in interphase chromosomal domain formation and in enhancer-promoter interactions. Instead, we suggest that the previously reported effects of condensin perturbation may result from their structural role during mitosis, which might indirectly impact the re-establishment of interphase chromosomal architecture after cell division.

2019-05-05 | GSE122157 | GEO

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

Project description:The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin’s loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II’s NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1’s ability to block condensin II’s association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both cohesin and condensin must be tightly regulated to adjust the structure of chromatids for their successful segregation.

2021-11-19 | GSE188988 | GEO

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

Project description:MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

| PRJNA780949 | ENA

Genome-wide analysis of condensin binding sites in vertebrate genome

Project description:The condensin complex is well known to be essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes. To date, the genome wide location and the nature of condensin binding sites has remained elusive in vertebrate systems. A detailed knowledge of condensin binding sites is essential to understand the as-yet enigmatic function of this complex and to define its greater role in chromosome architecture. To address this, we have used next generation sequencing to map condensin I in chicken DT40 cells. Unexpectedly, we found condensin I binds predominately to promoter sequences in mitotic cells. We also found a striking enrichment at both centromeres and telomeres, highlighting the importance of the complex in chromosome segregation. Taken together, the results show condensin I is largely absent from heterochromatic regions. These results reveal for the first time the condensin I binding sites in the vertebrate genome, and demonstrate the importance of the complex in genome segregation and suggest a novel function in gene regulation. Condensin associated DNA from chicken DT40 cells were affinity purified using streptavidin and deep sequenced by Illumina Hiseq2000

2013-10-03 | E-GEOD-45552 | biostudies-arrayexpress

Interaction mapping of human condensin I and condensin II complexes using DSBU-cross-linking and mass spectrometry

Project description:Human condensin I and condensin II complexes play essential roles in chromatin condensation during the cell division process. Both complexes use ATP as the energy source to pack extended chromatin into the short form of chromosome. In order to understand the mechanism of their function, we performed cross-linking mass spectrometry (XL-MS) analyses of both complexes and mapped the inter-molecular and intra-molecular interactions of the subunits.

2020-05-25 | PXD017531 | Pride

Genome-wide distribution of the three condensin complexes in C. elegans

Project description:Condensins are multi-subunit protein complexes that regulate chromosome structure throughout cell-cycle. Metazoans contain two types of condensin complexes (I and II) with essential and distinct functions. In C. elegans a third type of condensin (IDC) functions as part of the X chromosome dosage compensation complex1,2. We mapped genome-wide binding sites of the three condensin types in C. elegans embryos. Characteristics of condensin binding are similar between condensin types. ChIP-seq profiles of C. elegans subunits of the three condensins in 3-6 replicates from mixed stage embryos, controls are included, and RNA-Seq profiles of C. elegans in 5 replicates from mixed staged embryos. Additionally, ChIP-seq profiles of the condensin II subunit KLE-2 in 6 replicates from L3 with controls, and RNA-Seq profiles of KLE-2 mutants in 3 replicates each from L3.

2013-09-16 | E-GEOD-45678 | biostudies-arrayexpress

Genome-wide binding of condensin II and TFIIIC upon respective knockdown of a TFIIIC and condensin II subunit

Project description:Condensin complexes are highly conserved for chromosome compaction to ensure their faithful segregation in mitosis. However, little is known about the role of condensin complexes in interphase. Condensins exists in two complexes, condensins I and II, in higher eukayotic cells. During interphase, condensin II is predominantly localized in the nucleus throughout the cell cycle, whereas condensin I is localized at the cytoplasm in interphase. The distinct localization patterns suggest that condensin II, but not condensin I, may contribute to genome organization in interphase. Our results suggest that condensin II is associated with TFIIIC complex in vivo. The aim of these experiments is to unravel the dependency of each other on binding to chromatin.

2017-06-01 | GSE80034 | GEO

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